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United States Patent |
6,150,094
|
Maier
,   et al.
|
November 21, 2000
|
Use of an osmolyte for reducing or abolishing no-covalent interactions
of biological molecules to inert surfaces
Abstract
The present invention relates to the use of an osmolyte for reducing or
abolishing non-covalent interactions of biological molecules to inert
surfaces. Furthermore, the present invention relates to kits that may be
employed for uses in accordance with the present invention.
Inventors:
|
Maier; Elmar (Berlin-Dahlem, DE);
Ivanov; Igor (Berlin, DE)
|
Assignee:
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Qiagen GmbH (Hilden, DE)
|
Appl. No.:
|
862984 |
Filed:
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May 23, 1997 |
Foreign Application Priority Data
Current U.S. Class: |
435/6; 435/7.1; 435/7.2; 435/7.92; 435/91.1; 435/91.2 |
Intern'l Class: |
C12Q 001/68; G01N 033/53; G01N 033/567; G01N 033/537 |
Field of Search: |
435/6,91.1,91.2,7.1,7.2,7.92
|
References Cited
U.S. Patent Documents
5181999 | Jan., 1993 | Wiktorowicz | 204/180.
|
5587128 | Dec., 1996 | Wilding et al. | 422/50.
|
Foreign Patent Documents |
0153875 | Sep., 1985 | EP | .
|
0292809 | Nov., 1988 | EP | .
|
0363091 | Apr., 1990 | EP | .
|
0669401 | Aug., 1995 | EP | .
|
4411588 C1 | Sep., 1995 | DE | .
|
WO 93/22058 | Nov., 1993 | WO | .
|
WO 95/20682 | Aug., 1995 | WO | .
|
Other References
Moncef Zouali, et al., "A Rapid ELISA For Measurement of Antibodies to
Nucleic Acid Antigens Using UV-treated Polystyrene Microplates," Journal
of Immunological Methods, 90:105-110 (1986).
Thakar, M., et al., "Osmolyte Mediation of T7 DNA Polymerase and Plasmid
DNA Stability," Biochemistry 33: 12255-12259 (1994).
|
Primary Examiner: Riley; Jezia
Attorney, Agent or Firm: Fish & Neave, Massaro, Esq.; Jane A., Shin; Elinor
Claims
What is claimed is:
1. A method for reducing or abolishing non-covalent interactions between an
inert surface and a biological molecule, comprising adding a zwitterionic
osmolyte of the structural formula:
##STR2##
wherein R1, R2, and R3 are each selected from the group consisting of H,
CH.sub.3, C.sub.2 H.sub.5 or any other alkyl, and R' is any amino acid
residue, prior to or simultaneously with adding the biological molecule.
2. The method according to claim 1 wherein said zwitterionic osmolyte is
selected from an amino acid or its methylation product.
3. The method according to claim 1 wherein said zwitterionic osmolyte is
selected from betaine or glycine-betaine.
4. The method according to any one of claims 1, 2 or 3 wherein said
osmolyte is present at a final concentration of 1 to 2.5 M.
5. The method according to any one of claims 1, 2 or 3 wherein said
biological molecule is a macromolecule.
6. The method according to claim 5 wherein said macromolecule is selected
from a carbohydrate, a polynucleic acid, a polypeptide or combinations or
modifications thereof.
7. The method according to any one of claims 1, 2 or 3 wherein said
biological molecule is selected from a peptide, an oligonucleotide or
combinations or modifications thereof.
8. The method according to claim 7 wherein said polynucleic acid or
oligonucleotide is DNA.
9. The method according to claim 7 wherein said polynucleic acid or
oligonucleotide is RNA.
10. The method according to claim 7 wherein said inert surface is a silicon
surface, a silicium wafer, a glass surface or combinations or chemical
modifications thereof.
11. A kit comprising
(a) a concentrated stock solution of an osmolyte as specified in any of the
preceeding claims;
(b) a reaction buffer formulation containing an osmolyte as specified in
any of the preceeding claims; and/or
(c) an enzyme formulation containing an osmolyte as specified in any of the
preceding claims.
12. A method for reducing or abolishing non-covalent interactions in a PCR
reaction between an inert surface and a biological molecule, comprising
adding a zwitterionic osmolyte of the structural formula:
##STR3##
wherein R1, R2, and R3 are each selected from the group consisting of H,
CH.sub.3, C.sub.2 H.sub.5 or any other alkyl, and R' is any amino acid
residue,
to the inert surface prior to or simultaneously with adding the biological
molecule.
13. The method according to claim 12, wherein the biological molecule is an
oligonucleotide.
14. The method according to claim 12, wherein the inert surface is a
silicon surface, a silicium wafer, a glass surface or combinations or
chemical modifications thereof.
15. The method according to claim 12, wherein the osmolyte is a betaine or
glycine-betaine.
16. The method according to claim 12, wherein said zwitterionic osmolyte is
selected from an amino acid or its methylation product.
17. The method according to any one of claims 12, 15 or 16 wherein the
osmolyte is present at a final concentration of 1 to 2.5 M.
Description
This application claims priority under 35 U.S.C. .sctn.119 from European
patent application Serial No. 96108278.1, filed May 23, 1996.
The present invention relates to the use of an osmolyte for reducing or
abolishing non-covalent interactions of biological molecules to inert
surfaces. Furthermore, the present invention relates to kits that may be
employed for uses in accordance with the present invention.
The development of new materials has had a significant impact on a wide
variety of modern technologies. For example, the introduction of e.g.
silicon, gallium arsenite or polycrystal materials has in the past
strongly propagated the semiconductor technology. In modern biology,
similar developments have been observed. Thus, over the past two decades,
materials have been established in immunometric methods such as ELISAs
that allow for a standardization of experimental protocols and a
minimization of material-based error-rates in experimental results. A
different but equally important development is concerned with the
promotion of novel carrier materials to be used in (column) chromatography
for the purification and isolation of biological compounds of interest.
One of the major troubles that scientists have faced in a large number of
biological analytical and isolation techniques is that experimental
accuracies and production yields may be impaired by background or
undesired and possibly unspecific interaction problems. Such problems can
arise, for example, by non-covalent binding interactions of proteins or
other biological compounds to carrier surfaces. In order to overcome such
problems in ELISA techniques, free binding sites on carrier materials such
as polystyrrol are "blocked" with unrelated biological materials such as
heterologous proteins prior to testing for the compound of interest.
A technology that has in the most recent past revolutionized molecular
biology is the PCR technology. Naturally, in view of the wide
applicability of PCR, major efforts have been and are presently undertaken
to further improve facettes of this technology. One of these efforts is
directed to the creation of microreaction volumes of PCR chips, i.e.
microfabricated silicon chips bonded to a piece of flat glass to form a
PCR reaction chamber; see, e.g., Shoffner et al., "Chip PCR. I. Surface
passivation of microfabricated silicon-glass chips for PCR", Nucl. Acids
Res. 24 (1996), 375-379. Since native silicon which so far has been used
in the production of PCR chips has turned out to be an inhibitor of the
PCR, these investigators searched for materials and methods to obtain
reliable PCR results unimpaired by such inhibition or background problems.
They propose to use an oxidized silicon (SiO.sub.2) surface to achieve
their goal.
In spite of these achievements, it would be highly desirous and
advantageous to find a way of reducing or eliminating non-specific or
non-covalent interactions between inert surfaces and biological molecules
without having to modify commonly commercially available surfaces or being
limited to the use of only a small selection of suitable inert surfaces. A
successful development of such a means would, of course, have a wide
applicability in modern biology and not be restricted to the use in the
PCR technology. For example, Volkmuth and Austin have designed a method
for the micro-elelectrophesis of DNA molecules (Volkmuth and Austin, "DNA
electrophoresis in microlithographic arrays", Nature 358 (1992), 600-602).
Such techniques could also benefit from the inclusion of osmolytes such as
betaine into the buffer systems.
Thus, the technical problem underlying the present invention was to
overcome the prior art problems detailed hereinabove and develop a system
that reduces or eliminates undesired interactions of biological molecules
with inert surfaces.
The solution to said technical problem is achieved by providing the
embodiments characterized in the claims.
Accordingly, the present invention relates to the use of an osmolyte for
reducing or abolishing non-covalent interactions of biological molecules
to inert surfaces. In accordance with the present invention, it has
surprisingly been found that the addition of an osmolyte in a suitable
concentration to a solution comprising a biological molecule will reduce,
if not abolish the non-covalent interaction of said molecule with said
surface.
Osmolytes (or osmotic solutes) are found in a wide variety of
water-stressed prokaryotic and eukaryotic organisms. The three types of
osmolyte systems found in all such organisms except for the halobacteria
are polyhydric alcohols (such as glycerol and saccharose), free amino
acids and their derivatives (such as taurine and .beta.-alanine), and
methylamines (e.g. trimethylamine-N-oxide (TMAO), betaine and sarcosine)
or combinations of methylamines and urea (see, for a review, Yancey et
al., "Living with water stress: evolution of osmolyte systems" Science 217
(1982), 1214-1222).
One of the major advantages of the present invention is that the simple
addition of an osmolyte to such a solution conveniently reduces or
abolishes the interaction of said molecules with a wide variety of inert
surfaces. The special design or selection of an adequate surface for a
specific experimental set-up or purpose is therefore no longer necessary,
A further advantage of the present invention is that it allows for the
simple design of a variety of previously crucial experiments and therefore
saves time and costs for the interested investigator.
The term "biological molecule" as used herein refers to organic molecules
which are part of an organism or a living cell or derivatives of such
molecules. These molecules may be of natural, synthetic or semisynthetic
origin.
As used herein, the term "inert" has the meaning of "having little or no
ability to chemically react". It therefore bears the same meaning as
inertness in connection with nitrogens which occurs uncombined in the
atmosphere.
In a preferred embodiment of the use of the present invention said osmolyte
is a zwitterionic osmolyte.
In a most preferred embodiment of the use of the present invention said
zwitterionic osmolyte has the structural formula
##STR1##
wherein R.sub.1, R.sub.2 and R.sub.3 are H, CH.sub.3, C.sub.2 H.sub.5 or
any other alkyl, and R' is any amino acid residue.
In a further most preferred embodiment of the use of the present invention
said zwitterionic osmolyte is an amino acid or its methylation product.
In an additional most preferred embodiment of the use of the present
invention said zwitterionic osmolyte is betaine and preferably
glycine-betaine.
In a further preferred embodiment of the present invention said osmolyte is
present at a final concentration of 1 to 2.5 M. The advantageous
properties of said osmolyte also emerge, if it is included in the reaction
mixture at a lower concentration than 1 M. However, particularly
advantageous results are obtained, if the osmolyte is present in a final
concentration of 1 to 2.5 M.
In an additional preferred embodiment of the present invention said
biological molecule is a macromolecule.
The term "macromolecule" in connection with the term "biological molecule"
is perfectly clear to the person skilled in the art and need not be
described here any further.
In a most preferred embodiment of the use of the present invention, said
macromolecule is a carbohydrate, a polynucleic acid or a polypeptide, or
combinations or modifications thereof. Said combinations or modifications
need not necessarily have a biological function. In the alternative, their
biological function may not be known in the art (yet); see, for example,
the discussion about peptide nucleic acids (Nielsen et al., Science 254
(1991), 1497-1500) Yet, said modified biological macromolecules may have
essentially the same physico-chemical properties as the biological
macromolecules they are derived from and may find the same or similar
applications e.g. in molecular biology.
In a further preferred embodiment of the present invention said biological
molecule is a peptide or an oligonucleotide or combinations or
modifications thereof.
In a most preferred embodiment of the present invention said polynucleic
acid or oligonucleotide is DNA. The term "DNA" as used herein includes any
type of DNA, in particular cDNA and genomic DNA.
In a further most preferred embodiment of the present invention said
polynucleic acid or oligonucleotide is RNA. The term "RNA", as used in the
context of the present invention, is intended to mean any type of RNA and
in particular mRNA.
In a further preferred embodiment of the present invention said inert
surface is a silicon surface, a silicium wafer, a glass surface or
combinations or chemical modifications thereof.
Most conveniently, said substance is a manufactured silicon. Said silicon
may be obtained e.g., by standard manufacturing or processing techniques
such as photolithography.
The present invention additionally relates to a kit comprising at least
(a) a concentrated stock solution of an osmolyte as specified herein
before;
(b) a reaction buffer formulation containing an osmolyte as specified
herein before; and/or
(c) an enzyme formulation containing an osmolyte as specified herein
before.
The various components of the kit of the present invention are preferably
formulated in standard reaction vials and independently of one another.
The concentrations used in the stock solutions comprised in the kit of the
invention are suitable to allow an appropriate dilution of the osmolyte to
be useful in the teachings of the present invention. In the embodiments b)
and c) of the buffer of the invention, the osmolyte is preferably
contained therein in its final concentration. The ranges and limits of
said final concentrations have been provided herein before in the
specification.
The figures show:
FIG. 1: PCR In the presence of silicon particles and zwitterionic osmolytes
PCRs were carried out on a 1.2 kb fragment of human genomic DNA in the
presence of silicon particles (approx: 0.5 mm.times.0.5 mm.times.0.3 mm)
and 1 M betaine.
Lane 1: .lambda.-BstEll marker; lanes 2-5: PCR (50 .mu.l) carried out in
buffer with water and different amounts of silicon particles: lane 2: 10
particles; lane 3: 50 particles; lane 4: 75 particles; lane 5: no
particles as a control; lanes 6-9: PCRs (50 .mu.l) carried out in buffer
with 1 M betaine and different amounts of silicon particles: lane 6: 10
particles; lane 7: 50 particles; lane 8: 75 particles; lane 9: no
particles as a control.
FIG. 2: PCR in the presence of silicon powder and zwitterionic osmolytes
PCRs were carried out on a 1.5 kb fragment of human genomic DNA in the
presence of silicon powder and molar concentrations of betaine. Note that
the PCRs in molar concentrations of betaine allow efficient and specific
PCR amplification and reduce the inhibiting effect of silicon surfaces.
Lane 1: .lambda.-BstEll marker; lane 2: PCR carried out in a standard PCR
buffer without any silicon powder; lanes 3-6: PCR reaction carried out
with 4.6 mg of silicon powder in 100 .mu.l PCR volume; lane 3: water based
buffer; lane 4: 0.5 M betaine; lane 5: 1 M betaine; lane 6: 2 M betaine.
The examples illustrate the invention.
EXAMPLE 1
PCR in the Presence of Silicon Particles and Zwitterionic Osmolytes
In this example it is shown that the inhibiting effect of inert surfaces
such as pure silicon surfaces in a PCR as a nucleic acid template
dependent reaction can be reduced with the presence of osmolytes such as
betaine (in the examples glycine-betaine from Sigma was used).
Silicon is a very interesting material not only for the semiconductor
technology but also for the biological field, since it is a material which
can be easily handled and three dimensional micro-structures can be
manufactured very rapidly. Therefore, experiments such as PCR in the
presence of pure silicon are very important with a view of miniaturising
biological processes on e.g. silicon wafers in the future. Pure silicon is
generally known to inhibit biological processes such as PCRs for many
reasons. One of these reasons is that the molecules interact with
relatively large surfaces compared to the small volumes used in e.g. micro
reaction chambers. To test the inhibiting effects of pure silicon surfaces
in a PCR, PCR experiments on a 1.2 kb fragment of human genomic DNA cloned
in a M13 vector in the presence of crushed silicon particles (approx: 0.5
mm.times.0.5 mm.times.0.3 mm) as a model system were carried out.
The reactions were prepared with 1 ng of M13 DNA in 1.times. buffer (10 mM
Tris-HCl, pH 8.8 and 50 mM KCl) with increasing amounts (10, 50, 75) of
silicon particles in water (FIG. 1, lanes 2-5) and 1 M betaine (FIG. 1,
lanes 6-9)), 0.2 .mu.M of each primer (M13-40 (24-mer):
5'-CGCCAGGGTTTTCCCAGTCACGAC-3'; (SEQ ID NO:1); M13-Rev (24-mer):
5'-TTTCACACAGGAAACAGCTATGAC-3'), (SEQ ID NO:2) 100 .mu.M of each dNTP, 1.5
mM MgCl.sub.2 and 2.5 Units of Taq DNA polymerase in a total volume of 50
.mu.l. Reaction mixtures containing betaine were prepared by adding the
required mixture of water and a 5 M stock solution of betaine to the final
reaction volume of 50 .mu.l.
The mixtures were cycled 30 times at 94.degree. C. for 20 sec, 55.degree.
C. for 30 sec and at 73.degree. C. for 2 min 30 sec. 1 .mu.g of
.lambda.-BstEll digest was loaded on the gel as a marker. Further, 5 .mu.l
of the PCR products together with 2 .mu.l gel loading solution (70%
glycerol, 0.02 mg/ml bromphenol blue) were loaded on the agarose gels. The
results (see FIG. 1) show that the inhibiting effect of increasing amounts
of silicon particles can be overcome by adding betaine in molar
concentrations to the PCR buffer.
EXAMPLE 2
PCR in the Presence of Silicon Powder and Zwitterionic Osmolytes
In this example it is shown that the inhibiting effect of silicon powder in
a PCR that was shown recently (Shoffner et al., loc. cit.) can be
efficiently reduced by the presence of osmolytes such as the zwitterionic
osmolyte betaine in the reaction buffer.
PCR experiments were carried out using a 1.5 kb fragment of human genomic
DNA cloned in a M13 vector as a model system. The PCR with silicon were
prepared with 4.6 mg of silicon powder (Sigma, 325 mesh, 99%, no.
21,561-9) in a final volume of 100 .mu.l. This concentration of pure
silicon powder was recently shown to inhibit PCRs (Shoffner et al., loc.
cit).
PCRs were carried out in 1.times. buffer (10 mM Tris-HCl, pH 8.8 and 50 mM
KCl) in water only (FIG. 2, lane 2) and 4.6 mg of silicon powder (FIG. 2,
lanes 3-8) in water (FIG. 2, lanes 3), 0.5 M betaine (FIG. 2, lane 4), 1 M
betaine (FIG. 2, lane 5), 2 M betaine (FIG. 2, lane 6), 1 ng M13 DNA, 0.3
.mu.M of each primer (M13-40 (24-mer): 5'-CGCCAGGGTTTTCCCAGTCACGAC-3';
(SEQ ID NO:1) M13-Rev (24-mer): 5'-TTTCACACAGGAAACAGCTATGAC-3'), (SEQ ID
NO:2) (200 .mu.M of each dNTP, 1.5 mM MgCl.sub.2 and 10 Units of Taq DNA
polymerase in a total volume of 100 .mu.l. Reaction mixtures containing
betaine were prepared by adding the required mixture of water and a 5 M
stock solution of betaine to the final reaction volume of 100 .mu.l.
The mixtures were cycled 30 times at 94.degree. C. for 20 sec, 55.degree.
C. for 30 sec and at 73.degree. C. for 2 min 30 sec. 1 .mu.g of
.lambda.-BstEll digest was loaded on the gel as a marker. Further, 5 .mu.l
of the PCR products together with 2 .mu.l gel loading solution (70%
glycerol, 0.02 mg/ml bromphenol blue) were loaded on the agarose gels.
__________________________________________________________________________
# SEQUENCE LISTING
- - - - <160> NUMBER OF SEQ ID NOS: 2
- - <210> SEQ ID NO 1
<211> LENGTH: 24
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial - #Sequence: Synthetic
- # DNA
- - <400> SEQUENCE: 1
- - cgccagggtt ttcccagtca cgac - # - #
24
- - - - <210> SEQ ID NO 2
<211> LENGTH: 24
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial - #Sequence: Synthetic
- # DNA
- - <400> SEQUENCE: 2
- - tttcacacag gaaacagcta tgac - # - #
24
__________________________________________________________________________
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